was - University of Toronto...56 ZING YANG KUO AND TSI CHEN R. SHEN membrane is, in most cases, -due tlo improper position of the embryo, especially that of the beak. In such cases,

ONTOGENY OF EMBRYONIC BEHAVIOR IN AVES

XT. R.ERI>IRA*I,ION I N THE CHICK EMBRYO

ZING YANG KUO

Psychological Laboratory, University of Rochester

AND

TSI CHEN R. SHEN I

l3iologicaE Laborator.y, National University of Chekiang, Hangchow, China

llcceived for publiention, October 24, 1836

Intra-uterine respiratory movements in ma.mmalian foetus have been observed by many investigators. As early as 1800 Ahlfeld stated that certain rhythmic but, irregular intra-uterine movements of human foetus appeared to be due to the contraction of the respiratory muscles. He succeeded in obtaining graphic records, showing that when the foetal chest was expanded its abdomen contracted. Cohnstein and Zuntz ('84) observed that respiratory movements of the foetal lamb could be elicited by clamping of the umbilical cords. Rosenfeld and Snyder ('36) experimenti~ig on the rabbit foetus found that hyperventilation of the mother will cause the slowing of respiration or even apnoea in the foetus and that several minutes after suspension of artificial respiration, the foetus will resume its original respiratory rate. All tllcse observations seem to demonstrate that carbon dioxide tension is an essential factor for foetal respiratory movements. Pfliiger ('68) maintained that cutaneous stimulation by cold air is necessary in initiating respiratory movements in the foetal dog. Corey ('32) working on the foetal rat found that when delivered from the uterus in air and with foetal circulation left intact, respiratory movements occurred within 109 seconds, and that no reaction was obt.ained when the same procedure was carried out in isotonic saline solution. According to Corey, the initial respiratory movements are normally brought .?bout by increased

49

50 ZING YANG KUO AND TSI CHEN R. SHEN

CO, tension in the blood but the drying of the skin may have additional stimulating effect.

The respiratory function of the avian embryo has received very little attention. In the former articles of this series on the on- togeny of embryonic behavior in Aves (Kuo, '32a.) b, and c), the senior author has mentioned on several occasions that respiratory movements in the chick began two or three days before hatching, a.nd that in most cases irregular respiratory movements appeared many hours before the beak pierced through the membranes, and he has especially emphasized that such movements are of paramount importance to the process of hatching. The followirig rcport ~ i v c s t,llc rcsrlll,~ nntl grn,phic: rclc:orcls of o~ i r rcccnl; systctn- tttic study o ~ l tlie respirtbl.ory ~~lovctncrlts o f tJle cl1ic:lc crilbryo.

METHODS

Chick embryos from cggs of white Lcghonis were used in this experiment. The respiratory movements were studied by direct observation a.s well ns by kymograph records. Direct observa- tiori of tllc ~ ~ ~ o v c n l c ~ i l ~ s in t . 1 ) ~ ii~l.nc:l, egg wns Fltc:ilit.:a(,ctl by tho method of coating with vaseline the inner shell membrane (I<uo, '32a), so that the membrane was rendered transparent and the I-espiratory movements of the embryo could be easily observed. For the purpose of taking kymogrsph tracings, the whole embryo was shelled out from the egg, the amnion being cut. Great care wns takcn to injure RS little as possi1)le the cxtra embryonic circu- lation. The alii~nal w:is thcn carefully fastcncd to a 1)onrcl in such a way as to make it lie on its back beside the shell to which the blood vessels of the extra crnbryonic circulation werc pnr- tially attached. Uy means of a thread, the skin a t the anterior end of the keel of the thorax was connected with the shorter arm of the writing lever, and the respiratory movements 1ve1.e thus recorded on t,lle kymograph. (The do~vnstrokes in thc kytno- graph tracings represent inspiratory movements.) The younger embryos were shelled out and laid in t:he Petri dish instead of being fastened to :L board, tl~eir wings ant1 legs were loosely fixecl by cords which were clamped by haemostats. The body of the rnil~ryo wns Frequently ~noistc~lctl I)y cnot.torl p : ~ c b k s sonlic(l ill w:trrn

ONTOQENY O F EMBRYONIC BEHAVIOR IN AVES 61

isotonic saline solution. At the conclusion of each experiment the body length from the top of the beak to the tail end was measured. The temperature of the chamber in which the experi- ments were performed was raised to about 33°C.

RESULTS

The typical graphic records of respiratory movements in chicks from seventeen days of incubation to one day after hatching are reproduced in figure 1. The average frequency and the average amplitude of respiratory movements in several groups of chicks are given in table 1.

It was very difficult with the present technic to secure graphic rc(:ol'd~ for crrll)t.yv~ J(!HR than scvcntccn dnys of incubation, because the tissues were too delicate and fragile to be connected to the writing lever. Hence, we had to be content with direct observation by unaided eyes.

Corey ('32) has regarded the opening of the mouth in a gasping manner as an indication of true respiratory attempt. But in the cnsc of the chick embryo we considered as positive attempt a t active respiratory movcmcnts only the enlargements and depres- sions of the thoracico-abdominal cavities for the following rea- sons: (1) the opening and closing of the beak and swallowing movements occurred rather early in incubation (Kuo, '32a). Such movements could hardly be distinguished from gasping reflex in the later stage of incubation. (2) I t had been observed that rhythmic rnovemcnta of the thorax and abdomen were often times not accompanied by the opening of the beak.

Immediately after shelling out from the egg, embryos of 15 to 16 days of incubation were generally in an apnoeic condition, but after a few minutes of exposure in the air, slow and weak respiratory movements began to be observed; often times they were irregular and periodical. Gradually the rate of reupiration was increased, and the respiratory movements became more regular. .Respiratory movements, however, could not be ob- served in embryos younger than 16 days of incubation if they were not delivered out of the shell. But our observations seem to be sufficient to show that crnlx-yos of 3.5 to 16 dnys of incubation

54 ZING YANG KUO AND TSI CHEN R. SHEN

curve D the graphic records are similar to the Cheyne-Stokes' type of breathing. Our results agree with the findings of Salm and Vuori ('30). These investigators report that Cheyne-Stokes type of respiration frequently occurs in weak, premature infants, alternating with normal, regular breathing, and that it appears mostly in sleep, during rcstlcss periods and after warm baths, ctc.

Great individual variations in respirntion rate and amplilude were observed, but on average, both rate and amplitude are increased with the increase in the age of the embryo. (See table 1.) The difference in the rate between the embryos of 17 and 18 days of incubation on the one hand, and those of 20 and 21 days on the other, is very striking. The difference also holds good for amplitude. The increase in the rate of respiration during the last two days of incubation may probably be related to muscular activities in connection with hatching process.

It must be noted in passing that the averaged rates of respira- tion presented in table 1 which were taken after the embryos were taken out from the shell were slower than the readings (not reported in this paper) taken when the embryos were observed in the intact egg.

STAGE8 O F THE DEVELOPMENT O F RESPIRATORY MOVEMENTS

IN THE CHICK

Based on the observation of the intact egg through the trans- parent inner shell membrane the development of respiratory movelnent in tlic chick clnbryo lnny bc rouglily clnssificcl irdo three stages.' In stage one the muscular movement of the thoracic region is nlmost always accompnnied by movemcnts of other parts of the body, especially those of the trunk. In the 14 or 15 days embryo we found rising and depression of the thoracic region. But such motions can hardly be considered as true respiratory m0vement.s in view of the fact that they occur only as in integral part of the total bodily movement. Neverthe-

As a matter of fact, the movements of the thoracic region and the gasping reflex may be traced back to the early beak and neck, especially swallowing movemenfe and the movements of the trunk which are observed rather early in incubntion.

ONTOGENY O F EMBRYONIC BEHAVIOR I N AVES 55

less, the muscles involved in the rising and depression of the thoracic region are the same nluscles thaL are ialer employed in true respiratory function. In fact, the activities of the thoracic muscles in this stage are the primordial movements for future actual respiration.

In tlic sccontl sl.ago, wl1ic.h gcrlcrnlly occurs between the 17th and 18th tll~y of ir~(-l~l):~l,ior~, ir~(lc~)cn(Ic~~t rnovcrncnt~~ of the thoracic region, that is, movements not accompanied by other parts of the body, can be clearly observed. Such movements, which, as a matter of fact, are merely a transformation from the thoracic movements of the previous stage, may be regarded as true respiratory motions, although even in this stage very little or no air is inhaled or expellrd as the embryo is still surrounded by n liquid medium.

True pulmonary respiration takes place in the last or third stage which commences from the 19th or 20th day of incubation. During this stage, the amniotic fluid surrounding the beak of the embryo has already been dried up, and, in a great many cases, truc air-brcatliing can be obscrved cvcn hcfore the embryo h,w: torn up Llic shcll rncrnbranc, a fact which contradicts the state- ment made in most textbooks of embryology that the chick begins its first air breathing after the shell membranes have been punched. Careful observations have shown that during expira- tion the intact inner shell membrane is forced to bulge out appar- ently by thc air coming out from the nostril of the chick whilc 1 i i s i i o , t I I I I I I I ~ I I is j r s l . l'hc I~cginning of the tliirtl stage is a sure sign calling for extra oxygen for the 1irc~c1 of t l ~ r hatching ('hiel<. For the blood vessels of the extrn rml,ryonic circulalio~i are becornirig drgenerated so that the blood stream in the allantois can not carry in sufficient oxygen to supply the increasing need of the chick. Nor is the little air which may be present in t11c :trnnioLic cavity after the amniotic fluid is dricd up ant1 which may be breathctl by the chick enough to make up the deficit. And so, unless the embryo soon succeeds ill piinching throl~gh thc inner shell membrane and breathes the extra air contained in the air chamber, it invariably dies in the shcll i n n short fimc. (Fnil111.c of thrt cmhryo to pirrrc the shcll

C

56 ZING YANG KUO AND TSI CHEN R. SHEN

membrane is, in most cases, -due tlo improper position of the embryo, especially that of the beak. In such cases, the life of the embryo can be saved by artificially punching a hole through tlie membrane to allow air to go inside for the chick to breath. See Kuo, E and F.)

I t must be borne in ~nind thnt in the dcvelopn~ent of respirn- i ory funclio~i, tlie transitioli from o~ic stngc to another is gr:~dual and oftentime imperceptible. Furthermore, overlapping between I.wo slt~gcs is .in rr~lc rntl~cr ~II:LII rtn cxccl)lion.

The fact that Cop tension is an essent.ia1 factor in iniliating pulmonary respiration in the mammalian fetus has been empha- sized by previous i~~vcstigntors. This 1,rol)nbly holds good, also, for the chick. Ncctlhnm ('31, 1). 271) points o ~ i t that Ishc orlL~>llt of carbon dioxide fro111 the egg increases more than fifty times during incubation. (The increase is most rapid in the latter part of incubation.) Our observation of the changes in the color of I~lood in the extra.-embryonic circulations tends to confirm this view. From the 15th or 16th day of incubation on, the color of the blood vessels which cnn 1)c obscrvctl lllrough t,he inner shell membrane rendered l,ransparcnl by the coating of vaseline becomes darker and da.rker day after day. The work of Ham- mon and Zoll, also, indirectly lends support to the notion of rapid increase of CO, tension during the last few days of incubation. They found that the vitelline vessels are very sensitive to carbon tiioxide and that the latter has invariable constrictor cffect on thc vitelline vessels. Thcy thought tllnt :l.t t,hc cntl of incnl)al,ion, tlie large amount of COz accumulated constrictcd the vitellinc vesscls to such an cxtcnt ns to cause atrophy.

In this connrct,ion Irc are i~iclinetl to agree wit11 Corey tllat l>esides COz tension drying of the skin may serve as an additional factor in promoting respiratory movement~s. For we have found t;lirtt true respiratory rnovcments do not occur until tlle nlnriiotic liquid has been drictl. Furtliermore, : L I ~ injct-lion of 1 to 2 cc. of ivarm isotonic solut.ion into tlie dry amnion cavity in the front, of the beak of a. 20-dny embryo which was actively breathing tvo~ild definitely rcrl11c:e thr! rate of rcspirntion.

ONTOGENY O F EMBRYONIC BEHAVIOR IN AVES 57

DISCUSSION

The facts presented in the foregoing pages tend to further verify the view long held by the senior author that the development of behavior as well as physiological functions, is a gradual and continuous process. There is no abrupt or sudden change in any physiological function, either before or after birth. In the case of respiration in the chick not only has the contraction of the thoracic muscles a long and continuous history of development in embryonic life, but, also, the air-breathing activity comes about gradually. Moreover, the transition from allantoic to pulmonary respiration by no means suggests any abrupt change. While breathing is established, rtllantoic circlllation still remains. The cessation of the extra-embryonic circulation and the atrophy of the blood vessels have been observed to be a slow and gradual process.

SUMMARY

The development of respiratory movements in the embryonic chick was studied by graphic recording and by observation through tlie transparent shell mcrnbrane. The frequency and amplitude of respiration are presented in graphic form and in a table.

The development of respiratory movement in the chick embryo is divided into three stages. The main features in each stage and the transition from one stage to another have been described. Great stress is laid upon the gradualness and continuity in the ontogeny of various aspects of respiratory function.

COz tension is regarded as an important factor in stimulating pulmonary respiration. But the authors agree with Corey that drying of the skin has an additional stimulating value.

REFERENCES

AHLFELD 1890 Feutachrift Ftir Ludwig, Mnrhurg. COHNSTEIN, J., AND ZVNTZ, N.: 1884 Unteruuchungen tkher das Blut, den Kreis-

lauf und die Athmug beim SBugetheir-FBtus: Ptltkger'a Arch., 34, 173. COREY, E. L. 1932 Initin1 inspiration in the mammalian fetus. Jour. Exp.

Zool., 61, 1-12. Kno, Z. Y: 1932a Ontogeny of embryonic bel~avior in aves. I. The chronology